Program Director/Principal Investigator (Last, First, Middle). Tjian, Robert T. PROJECT SUMMARY (See instructions): During the remaining 5 year period of this R37 grant we propose to continue addressing several specific aims originally outlined includingdevelopment of defined in vitro transcription reactions with naked DNA and/or chromatin templates bearing endogenous promoters. We will complement these with single molecule analysisof PIC assembly and transcriptional initiation on single tethered DNA templates. We will continue our cell based and in vitro studies of the interplay between activators, repressers and co-factors. We will complement these with a focused study of singlecell analysis, particularly of transcription factor complexes and how they assemble and disassemble at transgenic promoter arrays or endogenous genes using fluorescently tagged transcription factors. A major ongoing focus will be to explore in greater detail our unexpected discovery that in terminally differentiated cells, there are major changes in the composition and function of the PIC components. Most striking was our finding that during skeletal muscle differentiation (myoblasts^myotubes) there is a wholesale loss of the prototypic TBP-TAF complex (TFIID) and its replacement by a novel smaller TRF3/TAF3 complex that is required for driving muscle specific transcription. We have now begun to assess whether similar dramatic remodeling of the PIC occurs in other terminally differentiated cell types including hepatocytes, motor neurons and mDA neurons. We will therefore devote a significant proportion of our effort in these next several years toward a more detailed analysis of this potentially new paradigm for regulating developmental cell fate and differentiation. Finally,we will continue our efforts to determine the structure-function relationships of activators interactingwith large multi-subunit components of the PIC including TFIID, CRSP/Med and RNA pol II by various structural methods including cryo-EM/single particle reconstruction as well as X-ray crystallography and low angle defraction. In summary, we believe that a combination of in vivo cell based studies, in vitro reconstitution reactions, single molecule assays and large particle structural determination taken in aggregate will provide us with the best opportunity to dissect the complex process of transcriptionalregulation in metazoan organisms. RELEVANCE (See instructions): This deeper insight into the molecular mechanisms of gene expression will in turn provide the basis for better understanding of disease states where dis-regulation leads to pathological states such as cancer, inflammation, diabetes and neurodegenerative diseases PROJECT/